Crystalline alkali metal salts of {60 -carboxybenzylpenicillin and process therefor

ABSTRACT

Crystalline mono-alkali metal and crystalline di-alkali metal salts of Alpha -carboxybenzylpenicillin and a process for their preparation from crude and amorphous forms of a Alpha carboxybenzylpenicillin and salts thereof, and from aqueous and organic solvent solutions of Alpha -carboxybenzylpenicillin and salts thereof.

United States Patent Butler [54] CRYSTALLINE ALKALI METAL SALTS OFa-CARBOXYBENZYLPENICILLIN AND PROCESS THEREFOR [63] Continuation-impartof Ser. No. 804,275, March 4, 1969, which is a continuation-in-part ofSer. No. 768,497, Oct. 17, 1968, abandoned.

[52] US. Cl ..260/239.l, 424/27] [51] Int. Cl. ..C07d 99/16 [58] Fieldof Search ..260/239.l

[451 Oct. 10,1972

[56] References Cited UNITED STATES PATENTS 3,142,673 7/1964 Hobbs et a]..260/239.l 3,282,926 11/1966 Brain et al. ..260/239.1

Primary ExaminerNicholas S. Rizzo Att0rneyConnolly and l-lutz [57]ABSTRACT Crystalline mono-alkali metal and crystalline di-alkali metalsalts of a-carboxybenzylpenicillin and a process for their preparationfrom crude and amorphous forms of a a-carboxybenzylpenicillin and saltsthereof, and from aqueous and organic solvent solutions ofa-carboxybenzylpenicillin and salts thereof.

13 Claims, No Drawings CRYSTALLINE ALKALI METAL SALTS or o-CARBOXYBENZYLPENICILLIN AND PROCESS THEREFOR CROSS-REFERENCE TO RELATEDAPPLICATIONS This application is a continuation-impart of my copendingapplication. Ser. No. 804,275, filed Mar. 4, l 969, which in turn is acontinuation-in-part of application, Ser. No. 768,497, filed Oct. l7,i968, now abandoned.

BACKGROUND or rm; INVENTION This invention relates to a novel,convenient, and economic process for producing crystalline, stable formsof a-carbosybenzylpenicillin. More particularly, it relates to theproduction of crystalline mono-alkali metal and crystalline di-alkalimetal salts of a-car'boxybenzylpenicillin.

The valuable broad spectrum penicillin, a-carbote ybenzylpenicillin, isdescribed in U.S. Pat. No. 3,l42,673 and in British Pat. l,004,670 alongwith'the preparation of various salts of this valuable penicillin,including the di-allrali metal salts. U.S. Pat. No. 3,l42,673 alsodescribed the preparation of the monoalkali metal salts. However, thealkali metal salts, as produced by the procedures of these references,are amorphous rather than crystalline materials.

The relatively high polar nature of a-carboxybenzylpenicillin-coupledwith its susceptibility to degradation, particularly at elevatedtemperatures and especially in alkaline solutions of about pH 9 orhigher or in acid solutions of about pH 4 or lower, to products such asbenzyl-penicillin, penicilloic acid, and penicillenic acid and thepresence of inorganic salts arising from the methods used for itspreparationrenders theforma' tion of its crystalline alkali metal saltsdifficult.

An amorphous salt is, in general, less desirable than is a crystallineform thereof from a preparative, sales, storage, and use standpoint. Thephysical propertiesof an amorphous salt, such as easeofhandling,.dispersion in solution, rate of solution, color of the salt,and so|ution thereof, are generally inferior to those of a crystallineform of the same salt. Additionally, amorphous forms of a salt arefrequently more hygroscopic than is a crystalline form of said salt.

For pharmaceutical usage, the above-mentioned inferior properties of anamorphous salt, relative to those of a crystalline form of the salt, areespecially detrimental. The formation of acceptable dosage forms, suchas solutions, suspensions, elixirs, tablets, capsules, and variouspharmaceuticaliy elegant preparations required and demanded by themedical and pharmaceutical professions, is rendered difficult orimpossible with an amorphous salt.

in the case of a-carboxybenzylpenicillin, the disadvantages of amorphousforms of its monoand di-allrali metal salts are of great concern sinceit is an extremely effective broad spectrum penicillin. The preparationof crystalline forms of its alkali salts and, particularly, of itssodium salt is, therefore, of vital interest to the pharmaceutical andmedical professions.

SUMMARY OF THE INVENTION It has been found that stable, crystallinefonns of the mono-alkali metal and di-alltali metal salts ofa-carboxybenzylpenicillin, including its epimers or stereoisomers, canbe conveniently prepared from aqueous or organic solvent solutions ofrr-carboxybeuzylpeuicillin andsalts thereof. and from crude andamorphous forms ofa carboxybe'nzylpenicillin and salts thereof, Theprocess utilizes readily available equipment and is characterized byease'of manipula tion, overall economy, and high quality of thecrystalline salts. The stable crystalline products of the presentinvention are useful in the samedosage forms and amounts for the samepurposes as are the amorphous prior art products but do not share theabove-mentioned shortcomings of the amorphous products.

By the term a-carboxybenzylpenicillin and salts thereof" is meant notonly the alkali metal salts, e.g., sodiumand potassium, but also thealkaline earth salts (calcium, magnesium, barium), the ammonium salt andsubstituted ammonium salt, e.g., procaine, dibenzyiamine,N,N'-dibenzyiethylenediamine, lephenamine, N-ethylpiperidine,diethylamine, triethylamine, and salts with other amines which havebeen; used to form salts with henzylpenicillin. it further includes themonoand disalts of a-csrboxybenzyl penicillin and its epimers. However,in view of the acidification step in the herein-described process whichconverts the above-mentioned salts to the acid form ofa-carboxybenzylpenicillin, all the salts are equivalent in this process.From a practical standpoint the crude and/or amorphous disodium salt ofmcarboxybenzylpenicillin is the usual starting material for thisprocess.

Alpha-carhoxybenzylpenicillin is highly susceptible to degradation,e.g., decarboxylation to benzylpenicil- [in and hydrolysis topenicilloic and penicillenic acids. Decarboxylation is readily broughtabout by heat. Hydrolysis occurs at both acid and alkaline pH levels andespecially at pH values above about 9 and below about 4. l-lydrolyticdegradation, of course, increases with an increase in temperature.

The process of this invention, therefore, achieves a balance as regardsthe above parameters of temperature and pH. Additionally, otherparameters, such as solubility factors and concentrations of reactants,are carefully woven into a unified process which conveniently affordsthe desired crystalline mono-alkali and di-alltali metal salts. By theterm alkali metal salts, as used herein, is meant the sodium andpotassium salts.

The process, in general, comprises a multiple extraction process whichpennits formation of concentrated solutions of a-carboxybenzylpenicillinwithout the use of heat and avoids the need for spray-dry or freeze-dryprocesses. A multiple extraction process need not be used if theconcentration of a-carborybenzylpenicillin or salts thereof in theaqueous solution is equivalent to at leastabout 5 percent (based ona-carboxybenzylpenicillin acid) concentration by weight. it is, however,desirable for concentrations less than about 5 percent by weight.

Preparation of the crystalline sodium and potassium salts ofa-carboxybenzylpenicillin from aqueous solutions of acarboxybenzylpenicillin and salts thereof. such as are produced by themethods of U.S. Pat. No.

3,142,673 and British Pat. l.004,670, or by alkaline hydrolysis of anaryl, e.g., phenyh-esterof a-carboxybenzylpenicillin. according to oneembodiment of the present invention, comprises:

a. adjusting the pH of an aqueous alkaline solution of '5-a-carboxybenzylpenicillin to about 2 to about 3.5,

b. extracting the acid solution-thus produced with a water-immiscibleorganic solvent,

c. back-extracting the waterrimmiscible organic solvent extractwithwater at a pl-l 'otfrom about 4.0 to about 8.0,

d. adjusting the pH of the aqueous extract thus produced to a value offrom about 2 to about 3.5, e.- extracting the aqueous acid solution thusproduced with a water-immiscible organicsolvent,

f. drying said water-immiscible .organic solvent extract to a watercontent of from about 3 percent to about 20 percent by. weight,

g. treating said dried extract with the proper proportion of an alkalimetal salt of an organic acid, such as potassium or sodium salts of2-ethyl hexanoic, caproic, oleic, ascorbic, glycolic, propionie,

acetic, succinic, cinnatnic, caprylic; and citric acids, or with sodiumor potassium hydroxide. methoxide, bicarbonate. or carbonate to providethe corresponding monoor di-alkali metal salt, and

h. separating the crystalline alkali metal salt.

An alternative, and favored embodiment of this invention, when theconcentration of o-carboxybenzylpenicillin or salts thereof in theaqueous solution is about 5 percent by weight or higher, comprisesproceeding directly from step (a) .to step (c) with elimination of steps(b) through (d).

DETAILED pascal-Prion on THE INVENTION The novel'processof thisinvention can, as mentioned above, be used to-prepare crystalline sodiumand potassium salts of a-carboxybenzylpenicillin from aqueous solutionsof a-carboxybenzylpcnicillin and salts thereof, and from crude and fromampboteric forms of a-carboxybenzylpenicillin and its salts.

it is desirable, for the purpose of this invention,-that the-aqueoussolution of o-carboxybenzylpenicillin or saltvthereof have aconcentration of a-carboxybenzylpenicillin equivalent to from about 0.5percent to about percent.of the acid from by weight. it is preferredthat the penicillin be present at a concentration of at least about 2percent by weight in order to achieve optirnum extraction .ofa-carboxybenzylpenioillin in subsequent steps.

The pH of the aqueous solution must be brought to a value of from about2.0 to about 3.5. The desired pH range is from about.2.0 to about 3.0;the favored range is from about 2.5 to about 3.0. The preferred range isfrom about 2.8 to about 3.0 since within this range degradation ofo'carboxybenzylpenicillin to o-carboxybenzylpenicilloic. acid isminimized. The pH adjustment is conveniently accomplished by means of amineral. acid, such as hydrochloric, sulfuric, and hydrobromic acids.The acid solution is then extracted with a water-immiscible organicsolvent, such as n-butanol, methyl isobutyl ketone, diethyl ether,diisopropyl-ether. The preferred solvents for this extraction aren-butanol and methyl isobutyl ketone in view of their favorablepartition coefficients for a-carboxybenzylpenicillin. Ethyl acetate andmethyl ethyl ketone can also be used but. because of their greatersolubility in water compared to that of the preferred solvent, recult inpoorer recovery yields. This loss in yield is corrected by adding highconcentrations of inorganic salts to the aqueous phase prior toextraction with these solvents. y

This extraction step can, of course, be conducted as a batch-wise orcontinuous process Irrespective of the type of process used, a totalvolume of solvent equal to about one-half to one-third the volume of theaqueous acid solution .has been found to permit satisfactory extractionof the, o-carboxybenzylpenicillin. Larger volumes can, of course, beused but necessitate the handling and recovery of unnecessary volumes ofsolvent. Smaller volumes are not desirable since the recovery of desiredproduct is decreased. The volume of organic solvent is advantageouslyregulated so as to provides concentration of 60 earboxybenzyipenicillinin the extract of from-about 5 percent to about 10 percent;

The water-immiscible organic solvent extract is then beck-extracted withwater at a pit of horn about 4.0 to about 8.0. The desirable pH. rangeis from about 6.5 to 8.0. The preferred range is from about pl-i 7.0 toabout 7.2. This particularstep affords opportunity to concentrate theu-carboxybenzylpenicillin into a small volume of solution and to thatfacilitate further processing. When the concentration ofo-carboxybenzyipenicillin, or its equivalent weight of sodium orpotassium salts. is about 5 percent or higher, this back-extraction isnot necessary as previously noted.

The aqueous extract isnext adjusted to pH of from about 2 to about 3.5with a mineral acid and preferably to a pl-lof from about2.2 to about3.0 and the acidified solution then extracted with a water-immiscibleorganic solvent such as these mentioned above. The preferredwater-immiscible organic solvents are n-butanol and methyl isobutylketone, in that order. When using methyl isobutyl itetoae as solvent, apH of about 2.2 is favored; and when using n-butanol, the favored pH isabout 3.0. It isdesirable, in this extraction step. to regulate thevolume of organic solvent so as to provide a concentration of from aboutlo-percent to about 20 percent of a-carboxybenzylpenicillinin the totalvolume of extract to facilitate subsequent precipitation of the desiredcrystalline salt.

The water-immiscible organic solvent extract ofacarboxybenzylpenicillin, of course, contains a certain amount of water.This wet' extract can be used. as such, in subsequent steps to producethe desired crystalline sodium or potassium salt ofo-carboxybenzyipenieillin. However, it has been observed that if toomuch water is present in the extract, that is, if it is too wet. the.crystalline product is difficult to filter, apparently becauseof a verysmall crystalsize. On the other hand, if the extract is too dry, thesalts tend to separate as amorphous rather than crystalline products.

It appears that a certain amount of water is highly desirable, in factnecessary, for optimum separation of the crystalline monoor di-alkalimetal salts. Too much water introducesmechanlcal problems and is,therefore, not desirable; and too little water tends to prevent or, atleast, minimize CW formation. The crystalline disodium salt, in fact,appears to be a hydrate.

It has been found that a water content of from about 3 percent to about20 percent in the organic solvent extract is conducive to crystalformation; the preferred range is from about 5 percent to about percentwater. This degree of dryness is readily and conveniently achieved bydrying the organic solvent extract with anhydrous sodium sulfate, thepreferred drying agent in this process. Other drying agents, such asanhydrous calcium sulfate and anhydrous magnesium sulfate, can also beused. When using magnesium sulfate, it is desirable to moniter thedrying so as to avoid dropping the water content below theaforementioned 3 percent level. Additionally, mechanical methods, suchas filtration through cotton, and azeotropic drying, can also be used.Further, the drying can conveniently be accomplished by the simpleexpedient of adding dry solvent, e.g., n-butanol, to the nbutanolextract. The volume of dry n-butanol to be added depends, of course,upon the degree of dryness desired. The addition of one volume of dryn-butanol to the n-butanol extract will, for example, lower the watercontent of the extract to one-half its value.

The dried organic solvent extract is then treated with the appropriatemolar proportion of the proper alkali metal salt of a suitable organicacid to form the desired crystalline monoor di-alkali metal salt ofacarboxybenzylpenicillin. Suitable salts are the alkali metal hydroxidesand alkoxides; e.g., methoxides; and the alkali metal salts of organicacids having from two to about eighteen carbon atoms such as thoseenumerated above and which are somewhat soluble in organic solvents suchas acetone, methyl isobutyl ketone, ethyl acetate, n-butanol,isopropanol, and/or in the water present in thea-carboxybenzylpenicillin containing extract. The presence of from about3 percent to about percent water in said extract thus serves a dualpurpose when using salts such as alkali metal acetates or hydroxides;i.e., as solvent and as a vital component of the crystallizing solution.The alkali metal salts of organic acids having from two to eight carbonatoms, such as acetic, butyric, caproic, and caprylic acids and thealkali methoxides are favored. The preferred organic acid salts aresodium and potassium Z-ethyl hexanoate because of their availability andadequate solubility in organic solvents.

The sodium or potassium Z-ethyl hexanoate, or other alkali metal salt,is desirably added in the form of a solution in acetone, methyl isobutylketone, ethyl acetate, n-butanol, isopropanol, or other organic solventin which it is soluble to at least about 0.05 percent. The preferredsolvent is nbutanol. When preparation of a crystalline mono-alkali metalsalt, e.g., the monosodium salt, is desired, up to one mole of sodiumZ-ethyl hexanoate, or other organic acid sodium salt, is used per moleof a-carboxybenzylpenicillin. A molar proportion much greater than lzlat this stage tends to reduce the yield of monosodium salt throughformation of the disodium salt. A molar proportion considerably lessthan lrl also results in reduced yields through incomplete reaction. Amolar ratio of from about 0.821 to about l.2:l is preferred for monosaltformation.

Formation of the crystalline di-alkali metal salts, e.g., the di-sodiumsalt, is achieved by using at least a 2:1 molar proportion of sodiumZ-ethyl hexanoate (or equivalent salt) to a-carboxybenzylpenicillin. Inactual practice, it is preferred to employ up to a 20 percent excess ofsodium Z-ethyl hexanoate (or equivalent salt) to ensure as completeprecipitation of the disodium salt as possible. Similar considerationsapply to the dipotassium salt.

The crystalline sodium or potassium salt (monoor disalt, depending onthe proportion of sodium or potassium 2-ethyl hexanoate or otherequivalent salt used) which precipitates is separated, e.g., byfiltration, then washed and repulped with an organic solvent such asacetone, isopropanol, or isopropyl ether, then dried.

The products are conveniently dried at about 35 to 65 C. Other methodscommonly used in the art are also applicable, as those skilled in theart will recognize, e.g., air-drying, drying under nitrogen, drying invacuo. Higher temperatures, while operative, require close surveillanceto avoid decomposition of the product and/or loss of water of hydration.A certain amount of water of hydration is desirable for stability of thecrystalline disodium salt.

The crystalline disodium salt thus obtained is somewhat hygroscopic butconsiderably less so than is the amorphous disodium salt. It appears tobe a monohydrate. The crystalline monosodium and monopotassium salts areconsiderably less soluble in water than are corresponding disodium anddi-potassium salts.

The temperature at which these various operations are conducted is notespecially critical. However, because of the instability ofa-carboxybenzylpenicillin in aqueous acid or aqueous alkaline solutionsit is advantageous to carry out the entire process, with the exceptionof the final drying step, at a temperature of from about 0 C. to about30 C., the preferred range from about 5 C. to about 25 C. The only stepof the process which can deviate from this range without detriment tothe product is the final step of drying the crystalline salts. Whenusing an alkali metal acetate or hydroxide as the neutralizing salt, itis desirable to operate at the upper limit of this temperature; e.g.,about 20-30 C. in order to achieve solubility of said salt in thea-carboxybenzylpenicillin containing extract.

Adaptation of the above-described sequence [steps (a) to (h)] to theconversion of crude and/or amorphous forms of a-carboxybenzylpenicillinand salts thereof to the crystalline monoor di-alkali metal saltsfollows the above sequence in part. It is, of course, obvious that ifone begins with an amorphous form of the acid, disodium salt, or othersalt such as the potassium salt, one need not begin at step (a) abovebut may begin at step (c), step (d) or step (e), depending on the form,e.g., acid or salt and purity of the material to be converted.

in addition to the above-described process, various alternativeextraction procedures are possible, as those skilled in the art willrecognize. One such sequence which affords the crystalline monoordi-alkali metal salts, and which is also applicable to aqueous reactionmixtures of a-carboxybenzylpenicillin and salts thereof produced by theprior art methods and especially to those produced by the alkalinehydrolysis of esters of acarboxybenzyl-penicillin, such asa-carbophenoxybenzylpenicillin, is a simplified version of theabovedescribed process. This sequence comprises, in general, adjustmentof the pH of the aqueous solution to a value of about 6 to about 7,followed by extraction of the neutral or essentially neutral solutionwith a water-immiscible organic solvent of the type mentioned in theabove sequence and preferably with n-butanol or methyl isobutyl ketone.The aqueous solution is next adjusted to a pH of from about 2 to about3.5 and extracted with n-butanol or methyl isobutyl ketone or otherwater-immiscible organic solvent as is mentioned above. The organicextract is then partially dried and treated with sodium and potassium2-ethyl hexanoate or other equivalent salt, as described above. The sameconditions and materials described in detail above apply in thisalternative sequence. As those skilled in the art will recognize, thedi-alkali metal salts of a-carboxybenzylpenicillin can be obtained byfurther neutralization of the mono-alkali metal salts. For this purpose,of course, the same, or a different, alkali metal salt used to preparethe mono-alkali metal salt can be employed. It is convenient to use analkali metal hydroxide or bicarbonate for this additionalneutralization.

Crystalline monosodium a-carboxybenzylpenicillin is, relative to theprior art described amorphous monosodium salt, of greatly improvedstability upon storage. Table l below presents the results of an accelerated stability study of the two salts. The procedure comprised storingsamples of the two salts at 65 C. for a period of l l days and analyzingthem at given intervals for their a-carboxybenzylpenicillin penicilloicacid content via the iodometric procedure set forth in the United StatesPharmacopeia XIV, 429. This assay procedure also detects anya-carboxybenzylpenicillin penicillenic acid present. The assay isreported in terms ofthe penicilloic acid content of the sample.

TABLE l.

Stability of Amorphous and Crystalline Monosodiuma-Carboxybenzylpenicillin at 65 C.

Carb. monosodium a-carboxybenzylpenicillin determined by the standardhydroxylamine assay for penicillins.

A second study of the stability of amorphous and crystalline mono-sodiuma-carboxybenzylpenieillin samples held at ambient temperature for 135days and assayed as mentioned above produced the following results(Table II):

TABLE ll.

Stability of Amorphous and Crystalline Monosodiuma-Carboxybenzylpenicillin Stored at Ambient Temperature for 135 daysAmorphous Salt Crystalline Salt Time Penicilloic Penicilloic (Days) AcidAcid (5) A comparative hygroscopicity study of amorphous and crystallinemonosodium a-carboxybenzylpeniciilin, conducted by storing samples ofthe salts in an atmosphere of 50 percent relative humidity at 25 C. andperiodically determining the increase in weight, showed the crystallinemonosodium salt to be far less hygroscopic than the amorphous monosodiumsalt (Table lll):

TABLE III.

Hygroscopicity of Amorphous and Crystalline Monosodiuma-Carboxybenzylpenicillin at 50% Crystalline Disodiuma-Carboxybenzylpenicillin from Crude Amorphous a-CarboxybenzylpenicillinA. Crude disodium a-carboxybenzylpenicillin (250 g.), prepared accordingto Example 3 of British Pat. 1,004,670 (see Preparation A), is dissolvedin water (2.5 liters), methyl isobutyl ketone (600 ml.) added, and themixture cooled to 5 C. The pH then adjusted to 2.2 by the addition, withstirring of 2N hydrochloric acid (approximately 650 ml.) over a 20-minute period. The mixture is thoroughly mixed and the methyl isobutylketone separated. The aqueous acid is extracted further with 2 X 600 ml.of methyl isobutyl ketone.

The extracts are combined, water l ,2 50 ml.) added, and the mixturecooled to 8 C. The pH is adjusted to 7.0 by addition of a saturatedaqueous solution of sodium bicarbonate (i600 ml.) and the aqueous phaseseparated. n-Butanol (650 ml.) is added to the aqueous phase which iscooled to 5-8 C. and adjusted to pH 3.0 by means of 2N hydrochloric acid(850 ml.). The mixture is thoroughly agitated, the n-butanol phaseseparated, and the aqueous phase extracted twice more with 400 and 250ml. volumes of n-butanol. The n-butanol extracts are combined, driedwith anhydrous sodium sulfate (600 g.), and filtered first throughfilter paper and then through a millipore filter unit (solvent resistantmembrane type, porosity 2.2

T0 the filtrate, there is then added sodium 2-ethyl hexanoate (2.1equivalents in 950 ml. acetone) over a l.5 hour period. Acetone is addedas needed to maintain an easily stirrable suspension (total volume addedis 2300 ml. Stirring is continued for one hour at room temperaturefollowing addition of the sodium 2-ethyl hexanoate, after which theproduct is removed by filtration. The filter cake is reslurried inacetone at room temperature for 45 minutes, then filtered, and thereslurry step again repeated. The crystalline disodium salt is driedovernight under a stream of nitrogen. Further drying is accomplished byheating the product to about 65 C. in a drying oven. Overall yield l78.5g., 71 percent.

B. The use of 1.05 equivalents of sodium 2-ethyl hexanoate in the abovesequence in place of 2.l equivalents produces the crystallinemono-sodium salt.

Repetition of procedures A and B above, but backextracting the methylisobutyl ketone solution of a-carboxybenzylpenicillin at pH values of4.0 and 6.5, rather than at pH 7.0, produces similar results.

EXAMPLE [I Crystalline Disodium a-Carboxybenzylpenicillin from Crudea-Carboxybenzylpenicillin A sample of crude a-carboxybenzylpenicillin(7.5 g.) is dissolved in water (75 ml.) to provide a l percent solutionof the crude acid and cooled to 10 C. The pH is adjusted to 2.2 by meansof 2N sulfuric acid, the solution extracted with methyl isobutyl ketone(3 X 20 ml.), and the combined extract dried with anhydrous calciumsulfate. The dried extract is filtered, cooled to 8 C., and treateddropwise with 18 ml. of an acetone solution of sodium 2-ethyl hexanoate(containing 0.2075 mg. of sodium salt per ml.). Acetone is added asneeded to permit efficient stirring. After l hour at 8-l0 C., thecrystalline disodium salt is removed by filtration, washed with acetone,and dried at 40 C.

Repitition of this procedure but using anhydrous magnesium sulfate,anhydrous sodium sulfate, centrifugation, filtration through cotton, orazeotropic drying in place of anhydrous calcium sulfate also producesthe crystalline disodium salt.

EXAMPLE 1]] Crystalline Disodium a-Carboxybenzylpenicillin from an Esterof a-Carboxybenzylpenicillin A. Phenylmalonic Acid Monophenyl EsterPhosphorous oxychloride (42l g.) and phenol (576 g.) are added tophenylmalonic acid (I000 g.) in a 3- neck, l2-liter flask. The contentsof the flask are mixed well and then heated on a steam bath for 4.5hours under an atmosphere of nitrogen. The temperature of the mixture isnot allowed to rise above 87 C. Under these conditions, the mixtureforms a clear melt within about [5 minutes, and evolution of hydrogenchloride is almost complete in 4 hours. Benzene (3.8 liters) is added tothe reaction vessel and the mixture refluxed for 45 minutes. The hotbenzene solution is decanted from the gummy phosphoric acid and phenolinto ice/water, the pH of the aqueous phase adjusted to 6.5 by additionof 8N sodium hydroxide, and finally to pH 7.8 by addition of 10 percentsodium carbonate solution. The mixture is mixed well and the aqueousextract collected. The benzene layer is washed with a small volume ofwater which is then combined with the first aqueous extract, and thecombined aqueous solution adjusted to pH 2.2 by addition of concentratedhydrochloric acid. The heavy precipitate produced is extracted intochloroform (total volume, 5 liters), the extract washed once with water,dried (anhydrous sodium sulfate), and evaporated to a solid whichcrystallizes when triturated with hexane. The product is filtered,washed with a small volume of 5 percent chloroform in hexane, andair-dried. Yield 430 g., m.p. l03-l 10 C. (33 percent). B. PhenylmalonylChloride Monophenyl Ester Thionyl chloride (92.5 g.) is added to asolution of phenylmalonic acid monophenyl ester in methylene chloride(1,000 ml.) and the solution refluxed gently for 4 hours. Toluene (50ml.) is added and the mixture evaporated under reduced pressure toprovide the desired product as a syrupy amber liquid. C.a-Carbophenoxybenzylpenicillin N-Ethylpiperidine Salt A solution of6-aminopenicillanic acid (86.4 g.) in water (850 ml.) is adjusted to pH6.7 by addition of 8N sodium hydroxide, then cooled to l0 C.Phenylmalonyl chloride monophenyl ester is added in one portion and themixture stirred vigorously for 40 minutes while the ph is maintained atpH 6.5-7.0 by addition of 2N sodium hydroxide. The cooling bath isremoved from the reaction vessel l0 minutes after addition of the acidchloride is complete. The reaction mixture is acidified (pH 2.l) byaddition of 2N hydrochloric acid and extracted with ethyl acetate. Thecombined ethyl acetate extracts are shaken with a 10 percent solution ofpotassium bicarbonate, the aqueous solution (pH 7.0-8.0) separated, andwashed once with a small volume of ethyl acetate which is discarded.Fresh ethyl acetate is added to the aqueous solution followed bysufficient potassium chloride to salt the product out of the aqueousphase. The aqueous layer is discarded and fresh water added to the ethylacetate solution. The potassium salt of the penicillin ester dissolvesreadily in the fresh water, and the ethyl acetate layer is discarded.The aqueous solution containing the desired product is acidified (pH2.2) with 2N hydrochloric acid, extracted with ethyl acetate, and theextract dried (Na SO,) and evaporated to provide 117 g. of crudea-carboxybenzylpenicillin phenyl ester. This material is redissolved inethyl acetate (l000 ml.) and 35.3 ml. of N- ethylpiperidine added. Theamine salt which crystallizes is collected by filtration and air-dried(97.0 g., 42.7 percent); m.p. l45l47 C. D. Hydrolysis ofa-Carbophenoxybenzylpenicillin N- Ethylpiperidine Salt Boric acid (6L8g.) and potassium chloride (74.56 g.) are dissolved in water andsufficient water added to the solution to bring the volume to 2 liters.A sufficient amount of 0.5N sodium hydroxide is added to adjust thesolution to pH 9.0. a-Carbophenoxybenzylpenicillin phenyl esterN-ethylpiperidine salt is dissolved in this buffer solution and themixture stirred at room temperature (25 C.) for 2.5 hours. The reactionmixture becomes more acidic as hydrolysis occurs until, at the cessationof reaction, the mixture is pH 8.45. The mixture becomes turbid duringthe last 15 minutes of the reaction due to separation of by-productphenol from the weakly basic solution. E. Isolation of CrystallineDisodium Salt The reaction is cooled to 0-10 C. and methyl isobutylketone (400 ml.) added, followed by sufficient 2N hydrochloric acid (ca.250 ml.) to acidify the mixture to pH 2.2. The mixture is allowed toseparate, the methyl isobutyl ketone extract collected, and the aqueousliquor extracted with two more volumes (each 300 ml.) of methyl isobutylketone. Water (200 ml.) is added to the combined extracts, the mixturecooled to 10 C., and a solution of sodium bicarbonate (8 g. in 20 ml. HO) added dropwise over a 5-minute period. To the mixture, now pH 5.8, asmall volume of saturated sodium bicarbonate solution is cautiouslyadded to bring the pH to 7.0. The aqueous extract ofa-carboxybenzylpenicillin sodium salt is collected and the methylisobutyl ketone liquor extracted with two separate volumes (150 ml.) ofwater. n-Butanol (100 ml.) is added to the combined aqueous extracts andthe mixture stirred and chilled to 0l0 C. Sufficient 2N hydrochloricacid is added to bring the mixture to pH 3.0, the mixture allowed toseparate, and the butanol layer collected. The acidic aqueous liquor isextracted twice with 75 ml. volumes of butanol, the combined butanolsolutions (250 ml.) dried over anhydrous sodium sulfate (300 g.) for onehour, then filtered into a 1,000 m1. 3-neck flask equipped with amechanical stirrer and an addition funnel. An acetone solution of sodium2- ethyl hexanoate (l69 ml. containing 207.5 mg. of salt per ml.) isadded dropwise over a 20 to 30-minute period. When approximatelyone-half of the solution has been added, the mixture is seeded withcrystalline disodium salt. The mixture is stirred for 30 minutesfollowing addition of the sodium ethyl hexanoate, then acetone (225 ml.)added, and the mixture stirred for an additional 15 minutes. Thecrystalline disodium salt is collected by filtration on a sintered glassfunnel under an atmosphere of dry nitrogen, and the filter cake washedwith acetone (500 ml.). The cake is slurried in fresh acetone (1000ml.), the slurry stirred at room temperature for 30 minutes, filtered asdescribed above, and allowed to dry on the filter in a stream of drynitrogen for 16 hours. The product is a white, freeflowing crystallinepowder, slightly hygroscopic and readily soluble in water.

Yield 25.6 g. (60 percent), m.p. l98-20lC. dec.

Repetition of the above procedure but using an nbutanol solution ofsodium 2-ethyl hexanoate in place of an acetone solution producessubstantially the same results.

EXAMPLE IV An aqueous hydrolysis mixture prepared according to theprocedure of Example Ill-D is adjusted to pH 7 by addition of 2Nhydrochloric acid, then extracted with n-butanol (3 X 200 ml.). Then-butanol extract is discarded. n-Butanol (100 ml.) is added to theaqueous phase, the mixture stirred, cooled to 5-l0 C., and brought to pH2.2 by means of 2N hydrochloric acid. The n-butanol phase is allowed toseparate, collected, and the aqueous layer extracted twice again withn-butanol (75 ml. each). The combined n-butanol extracts are dried overanhydrous sodium sulfate, filtered, and treated at 10 C. with sodium2-ethyl hexanoate (169 ml. of acetone solution containing 207.5 mg. ofsodium salt per ml.) over a 30-minute period. The mixture is stirred forone-half hour, 250 ml. acetone added, and stirring continued for anadditional minutes. The crystalline disodium salt is collected and driedunder nitrogen.

EXAMPLE v Repetition of the procedure of Example Ill-E but using 1.00equivalent of sodium 2-ethyl hexanoate in place of 2.10 equivalentsproduces the crystalline monosodium salt.

In like manner, the crystalline monoand di-potassiurn salts are preparedby the use of 1.00 and 2.20

equivalents of potassium 2-ethyl hexanoate in place of sodium 2-ethylhexanoate.

EXAMPLE VI The procedure of Example is repeated but using a pHadjustment to 3.3 in the initial acidification step. Substantially thesame results are obtained.

EXAMPLE Vll Crystalline Monosodium a-Carboxybenzylpenicillin Butanolsolutions of a-carboxybenzylpenicillin, prepared according to theprocedure of Example lll-A through Ill-E, are dried over anhydroussodium sulfate or magnesium sulfate as noted below then treated with anorganic solvent solution of a sodium salt to produce the monosodiumsalt.

I: water in n-butanol after drying molar equivalents ofsodium salt usedsufficient water is present in the n-butanol solution to completelydissolve the sodium acetate salt EXAMPLE Vlll Disodiuma-Carboxybenzylpenicillin Monosodium a-carboxybenzylpenicillin (0.01mole) prepared as in Example Vll is added to an aqueous solution ofsodium hydroxide (0.01 mole in 25 ml. water). The disodium salt isrecovered by freeze-drying.

Similarly the di'potassium salt is prepared from the mono-potassium saltof a-carboxybenzylpenicillin and potassium hydroxide.

The above-mentioned alkali metal hydroxides can, of course, be replacedby the corresponding alkali metal bicarbonates or carbonates withsubstantially the same results.

EXAMPLE lX Crystalline Mono-alkali Metal Salts ofa-Carboxybenzylpenicillin The procedure of Example Vll-(c) is repeatedbut using the following alkali metal salts in place of sodium 2-ethylhexanoate'.

sodium caproate sodium oleate sodium ascorbate sodium glycolate sodiumpropionate sodium succinate sodium cinnamate sodium caprylate potassiumcitrate potassium propionate potassium oleate potassium caproatepotassium ascorbate potassium glycolate potassium succinate potassiumcinnamate In each case the crystalline monosodium or potassium salt isproduced.

EXAMPLE X Crystalline Di-alkali Metal Salts of a-CarboxybenzylpenicillinThe procedure of Example [X is repeated but using 2.20 equivalents ofthe alkali metal salts enumerated therein per equivalent ofa-carboxybenzylpenicillin to produce the di-alkali metal salts.

EXAMPLE Xl EXAMPLE XII The procedure of Example VII-(h) is followed butsubstituting sodium and potassium methoxide in methyl isobutyl ketonefor sodium acetate trihydrate. In each instance, the mono-alkali metalsalt is produced.

The use of 2.0 equivalents of alkali metal methoxide produces thecorresponding di-alkali metal salt of acarboxybenzylpenicillin.

PREPARATION A Crude Disodium a-Carboxybenzylpenicillin (British Patent 1,004,670 Example 3) A mixture of phenylmalonic acid (25 g., 0.l40 mole)in dry ether (500 ml.), thionyl chloride (17.35 g., 0.]45 mole), and atrace of dimethyl-formamide is refluxed for 3 hours, then evaporatedunder reduced pressure at room temperature (finally by co-distillationwith dry benzene to remove the last trace of thionyl chloride).

The syrupy residue of mono acid chloride is dissolved in dry ether (300ml.) and added to a stirred, ice-cold mixture of 6-aminopenicillanicacid (30 g.), water (275 ml.), ether (150 ml.), and N sodium hydroxidesolution (l35 ml.). The mixture is stirred for 30 minutes, the aqueousphase adjusted to pH 2 by addition of dilute hydrochloric acid, and thelayers separated. The ether solution is washed with ice-cold water,saturated with ether (8 X 300 ml.), and then extracted with water towhich sufficient dilute sodium hydroxide has been added to bring theaqueous phase to pH 7. Evaporation of the aqueous solution at lowtemperature and pressure, followed by drying the residual white powderin a vacuum desiccator, gives the crude disodium salt ofa-carboxybenzylpenicillin (33.3 g.).

What is claimed is:

l. A process for making a crystalline alkali metal salt ofa-carboxybenzylpenicillin which comprises treating a solution ofa-carboxybenzylpenicillin in a solvent selected from the groupconsisting of n-butanol and methyl isobutyl ketone,

said solution containing from about 3 percent to about 20 percent byweight of water, with an alkali metal salt selected from the groupconsisting of a. an alkali metal salt of an organic acid selected fromthe group consisting of 2-ethyl hexanoic, caproic, oleic, ascorbic,glycolic, propionic, acetic, succinic, cinnamic, caprylic and citricacids; b. an alkali metal hydroxide; and c. an alkali metal methoxide,wherein the alkali metal is selected from the group consisting of sodiumand potassium.

2. The process of claim 1 wherein a molar ratio of an alkali metal saltof group (a) to a-carboxybenzylpenicillin of at least about l:l is usedand the crystalline alkali metal salt of a-carboxybenzylpenicillinseparated therefrom.

3. The process of claim 3 wherein the alkali metal salt is selected fromthe group consisting of an alkali metal salt of acetic acid and analkali metal salt of 2- ethyl hexanoic acid.

4. The process of claim 3 wherein an n-butanol solution ofa-carboxybenzylpenicillin containing at least about 5 percenta-carboxybenzylpenicillin by weight and from about 3 percent to about 15percent by weight of water is treated with at least a 2:1 molarproportion of sodium 2ethyl hexanoate in acetone and the crystallinedisodium salt is separated therefrom.

5. The process of claim 3 wherein an n-butanol solution ofa-carboxybenzylpenicillin containing at least about 5 percenta-carboxybenzyl-penicillin by weight and from about 3 percent to about15 percent by weight of water is treated with from about 0.8 to about1.2 molar proportion of sodium 2-ethyl hexanoate in nbutanol and thecrystalline monosodium salt is separated therefrom.

6. A process for making a crystalline alkali metal salt ofa-carboxybenzylpenicillin wherein the alkali metal is selected from thegroup consisting of sodium and potassium which comprises:

a. adjusting the pH of an aqueous alkaline solution ofa-carboxybenzylpenicillin to about 2 to about 3 .5,

b. extracting the aqueous acid solution thus produced with awater-immiscible organic solvent selected from the group consisting ofn-butanol and methyl isobutyl ketone,

c. drying said water-immiscible organic solvent extract to a watercontent of from about 3 percent to about 20 percent by weight,

d. treating said dried extract with an alkali metal 2- ethyl hexanoate,and

e. separating the crystalline alkali metal salt.

L or

solvent selected from the group consisting of acetone, ethyl acetate,n-butanol, and isopropanol.

11. A compound selected from the group consisting of the crystallinemonosodium salt and the crystalline monopotassium salt ofa-carboxybenzylpenicillin.

12. A compound selected from the group consisting of the crystallinedisodium salt and the crystalline dipotassium salt ofa-carboxybenzyl-penicillin.

13. The crystalline disodium salt of a-carboxybenzylpenicillin.

i i k

2. The process of claim 1 wherein a molar ratio of an alkali metal saltof group (a) to Alpha -carboxybenzylpenicillin of at least about 1:1 isused and the crystalline alkali metal salt of Alpha-carboxybenzylpenicillin separated therefrom.
 3. The process of claim 3wherein the alkali metal salt is selected from the group consisting ofan alkali metal salt of acetic acid and an alkali metal salt of 2-ethylhexanoic acid.
 4. The process of claim 3 wherein an n-butanol solutionof Alpha -carboxybenzylpenicillin containing at least about 5 percentAlpha -carboxybenzylpenicillin by weight and from about 3 percent toabout 15 percent by weight of water is treated with at least a 2:1 molarproportion of sodium 2-ethyl hexanoate in acetone and the crystallinedisodium salt is separated therefrom.
 5. The process of claim 3 whereinan n-butanol solution of Alpha -carboxybenzylpenicillin containing atleast about 5 percent Alpha -carboxybenzyl-penicillin by weight and fromabout 3 percent to about 15 percent by weight of water is treated withfrom about 0.8 to about 1.2 molar proportion of sodium 2-ethyl hexanoatein n-butanol and the crystalline monosodium salt is separated therefrom.6. A process for making a crystalline alkali metal salt of Alpha-carboxybenzylpenicillin wherein the alkali metal is selected from thegroup consisting of sodium and potassium which comprises: a. adjustingthe pH of an aqueous alkaline solution of Alpha -carboxybenzylpenicillinto about 2 to about 3.5, b. extracting the aqueous acid solution thusproduced with a water-immiscible organic solvent selected from the groupconsisting of n-butanol aNd methyl isobutyl ketone, c. drying saidwater-immiscible organic solvent extract to a water content of fromabout 3 percent to about 20 percent by weight, d. treating said driedextract with an alkali metal 2-ethyl hexanoate, and e. separating thecrystalline alkali metal salt.
 7. The process of claim 6 wherein theconcentration of Alpha -carboxy-benzylpenicillin in the aqueous alkalinesolution is from about 0.5 percent to about 20 percent by weight.
 8. Theprocess of claim 7 wherein the water-immiscible organic solvent ismethyl isobutyl ketone.
 9. The process of claim 7 wherein thewater-immiscible organic solvent is n-butanol.
 10. The process of claim7 wherein the dried n-butanol extract is treated with at least about a1:1 molar proportion of sodium 2-ethyl hexanoate dissolved in a solventselected from the group consisting of acetone, ethyl acetate, n-butanol,and isopropanol.
 11. A compound selected from the group consisting ofthe crystalline monosodium salt and the crystalline monopotassium saltof Alpha -carboxybenzylpenicillin.
 12. A compound selected from thegroup consisting of the crystalline disodium salt and the crystallinedipotassium salt of Alpha -carboxybenzyl-penicillin.
 13. The crystallinedisodium salt of Alpha -carboxybenzylpenicillin.